Our overall goal in this program project is to understand the potential of ligands of the human immunodeficiency virus (HIV-1) gp120 envelope glycoprotein to inhibit virus entry. HIV-1 infection is initiated by entry of the virus into host cells. The entry process depends on initial attachment of virus with host, and the molecular components of this attachment process are known. HIV-1 cell recognition occurs through envelope proteins on the viral spike, a heterotrimer of the gp120/gp41 complex. The interactions between gp120 and cell receptors appear to play the central role in viral entry. Hence, the design of antagonists of gp120 interactions with cell receptors is the central objective of the Program Project. We will utilize a comprehensive, interdisciplinary, multi-group approach that will combine chemical synthesis, high-resolution structure determination, thermodynamic and kinetic binding analyses, cell infection analyses, recombinant protein production and mutagenic analyses and computational modeling. We have already demonstrated our capacity to utilize this comprehensive approach as a team, and believe our experiences so far along with current starting points arrived at in our studies will successfully lead to important advances in mechanistic understanding and productive approaches to HIV-1 entry inhibitors. The Program Project will have 5 overarching specific aims. (1) Identify novel compounds that interact with functionally important regions of the HIV-1 gp120 glycoprotein. (2) Define the binding sites of the compounds on the gp120 glycoprotein. (3) Elucidate the mechanism of inhibition of HIV-1 entry associated with the compounds. (4) Optimize gp120-binding compounds for antiviral potency. (5) Understand the impact of variation in the HIV-1 envelope glycoproteins on the antiviral efficacy of the compounds. We will accomplish these aims through 5 Projects and 2 Cores. Project 1: Structure analysis of HIV-1 entry inhibition (Hendrickson); Project 2: Peptide-inspired competitive and allosteric inhibitors of HIV-1 entry (Chaiken); Project 3: Discovery and synthesis of small molecule CD4-gp120 antagonists (Smith); Project 4: Assembly and inhibition thermodynamics (Freire); Project 5: Structure-based antagonism of HIV-1 envelope function in cell entry (Sodroski); Core A: Computational modeling (LaLonde); Core B: Protein technologies (Chaiken). This program project will examine the potential of small molecules to inhibit HIV-1 viral entry and will identify candidate inhibitors as potential antagonists for AIDS prevention and treatment. Since many viral proteins involved in disease pathogenesis exhibit the type of plasticity found for HIV-1 gp120, we believe that the approaches we develop for the gp120 system will provide experimental paradigms for prevention and treatment of other diseases as well [unreadable] [unreadable] OVERALL PROGRAM EVALUATION: STRUCTURE-BASED ANTAGONISM OF HIV-1 ENVELOPE FUNCTION IN CELL ENTRY (PROGRAM DIRECTOR: CHAIKEN, IRWIN M., DREXEL UNIVERSITY) [unreadable] [unreadable] This is a renewal application submitted in response to the Program Announcement PA-07-30 entitled "Support of NIGMS Program Project Grants (P01)." The initial submission was responsive to RFA-GM-02-004 entitled "Structural Biology of AIDS Related Proteins." The initial application had four projects, three scientific cores and an academic core. In the renewal application, two of the previous cores - Crystallography core, led by Dr. Hendrickson, and the Biology core, led by Dr. Sodroski, have been upgraded as Projects 1 and 5, respectively. Project 1, Computational modeling (led by Dr. Head) has been replaced by Core A, which is now led by Dr. Lalonde. [unreadable] [unreadable] Project 1, led by Dr. Wayne Hendrickson, proposes to determine high resolution structure of HIV envelope protein and complexes in an attempt to develop mechanistic insights for design and analysis of entry inhibitors. Dr. Hendrickson is a highly accomplished X-ray crystallographer, and has been instrumental in solving the structure of HIV gp120, CD4 and their complex with the Fab fragment of monoclonal antibody (MAb) 17b, which have led to the discovery of series of potential inhibitors. A considerable progress has also made in the design of "mini" proteins whose structure mimic the CD4 receptor. The proposed research design is focused on mapping the conformational changes that occur in the structure of gp120 on recognizing CD4 that then direct the virus to chemokine receptors. Attempts to establish more details about the sequence of events that lead to cell infection could further enhance the value of these studies. Overall, this project was rated in the Excellent category with a score of 175. [unreadable] [unreadable] Project 2, led by Dr. Irwin Chaiken proposes to develop peptide-inspired competitive and allosteric inhibitors of HIV entry. Dr. Chaiken is an outstanding investigator with an excellent record of productivity. The proposed approaches are highly significant and innovative. While most studies of entry inhibitors have focused on competitive inhibition, this projects stands out with its emphasis on allosteric inhibition as well as competitive inhibition. In addition, the investigator will work on bifunctional inhibitors that bind to gp120 and block binding to 'both' CD4 and antibody 17b (a CCR5 surrogate). These approaches could provide unique insight into mechanisms of HIV inhibition, and possibly lead to the development of novel class of inhibitors. Despite the high enthusiasm for this proposal, the panel was slightly disappointed that none of the current inhibitors are particularly effective at virus inhibition. Overall, this project was rated in the Excellent category with a score of 170. [unreadable] [unreadable] Project 3, led by Dr. Amos Smith proposes the discovery and synthesis of small molecule CD4-gp120 antagonists. The expertise and productivity of the investigator are the major strengths of this application. This is a well designed and well written proposal and incorporates a number of innovative synthetic avenues. The "Click Chemistry" approaches, although interesting and highly innovative, are risky as it is unclear whether the gp120-CD4 complex will be in a sufficiently tight transitional state to yield useful results. The identification of mini-protein based agonists are particularly exciting, and may yield new insights in to the fusion process. The concept of dual site antagonists, although appealing, may prove to be problematic. Design of these studies could have been better developed. Overall, this proposal represents a good effort at dealing with an extremely challenging problem. Although it is disappointing that this Project has not generated any significant leads, investigations are on track towards rectifying this situation. Overall, this project was rated in the Excellent category with a score of 175. [unreadable] [unreadable] Project 4, led by Dr. Ernesto Freire, proposes thermodynamic studies of complex assembly and inhibition and hopes to provide information on the binding site of inhibitors, the level of ordering that occurs in gp120 upon binding the inhibitor, the binding affinity of the inhibitors, and the mechanism of binding of inhibitors (competitive or allosteric). These studies are highly significant and will help optimize inhibitor design. The proposed approaches are generally straightforward and Dr. Freire is a widely acknowledged expert in this area. The description of the project is rather brief. While the brevity of the proposal was not in itself a concern, inclusion of new experiments and description of new and relevant items like the structure of unliganded SIV gp120 would have been helpful. Overall, this project was rated in the Outstanding category with a score of 145. [unreadable] [unreadable] Project 5, led by Dr. Joseph Sodroski, proposes to test the effect of candidate compounds on the entry of HIV-1, carry out mechanistic studies of binding and inhibition of HIV-1 entry by the compounds, as well as investigate the impact of variation in the HIV-1 envelope glycoproteins on the antiviral efficacy of the compounds. The proposed investigations are highly significant, and the experimental approaches are well developed. Dr. Sodroski is an excellent investigator with an outstanding record of productivity. Overall, this project was rated in the Outstanding category with a score of 115. [unreadable] [unreadable] Core A is directed by Dr. Judith LaLonde and provides Computational Modeling support for the entire program. The description of the services offered, especially the technical details, have not been provided in adequate detail. There appears to be a considerable overlap between the computational efforts of Core A and the work described in Project 1. The qualification of the Core director was also hard to evaluate due to a paucity of publications, which is probably related to the fact that she was working in the industry till recently. The description of this Core was regarded to suffer from poor grantsmanship. Nevertheless the panel felt that the services offered by this core are important and necessary for the success of the program, and rated it as satisfactory. [unreadable] [unreadable] Core B is directed by Dr. Irwin Chaiken and focuses on protein technologies. This core will be responsible for the large scale production of various protein and peptide reagents and will supply these reagents, as needed, to all the other projects. The list of target proteins will be produced in quantity will include trimeric viral spike mimetics, mutants of CD4, anti-gp120 antibodies, and coreceptors CCR3, CCR5, and CXCR4. This core facility has effectively met the needs of the team to date, appropriate strategies are in place for large scale production of high-quality protein reagents. This core will likely play a dominant role in driving progress of this Program Project and was rated satisfactory. [unreadable] [unreadable] Overall, this program consists of comprehensive, interdisciplinary studies of structure-based antagonism of HIV envelope function in an attempt to design novel entry inhibitors. Five projects and two cores are proposed which are interdependent and complement each other well to achieve the stated objectives. The program director is highly accomplished and has efficiently managed the program, despite the fact that the different projects are distributed among different geographic locations namely - Drexel University (Philadelphia, PA), Columbia University (New York), Johns Hopkins University (Baltimore, MD), and Dana-Farber cancer institute (Boston MA). The proposed investigations are scientifically sound and address a medically significant topic. The progress has been excellent in the current funding period. The overall enthusiasm for this application was however dampened slightly by the concerns that these studies have not yet resulted in strong lead compounds despite the fact that the project is in its 12th year. [unreadable] [unreadable] [unreadable] PROJECT 1: STRUCURE AND ANALYSIS OF HIV-1 ENTRY INHIBITION (PROJECT LEADER: HENDRICKSON, WAYNE A., COLUMBIA UNIVERSITY) [unreadable] Numerical Rating: 175 [unreadable] [unreadable] DESCRIPTION (provided by applicant): This project is an investigation into the structural biology of HIV entry inhibition. Our overall objective is to provide an atomic-level understanding of opportunities for intervention in processes of cell entry by HIV. Continued work toward this goal will be conducted as Project 1 in the context of a Program Project on antagonism of HIV envelope function in cell entry. Our technical focus is on x-ray crystallography, so as to determine structures at sufficient resolution to develop mechanistic insights, but associated binding experiments and computations will also be performed. Our specific aims comprise structural analyses of complexes of HIV gp120 with chemically derivatized CD4, structural analyses of complexes of HIV gp120 with other candidate inhibitory molecules, structural studies of HIV gp120 interactions with other natural binding partners besides CD4, and innovative computational analyses of gp120 plasticity. We build not only on our recent experience in this program, but also on our earlier structural investigations of human CD4, HIV gp120s, and associated antibodies. We have adopted or developed both appropriate expression systems for making the required proteins and also appropriate assays for following inhibitory action. Interaction with other elements of the Program Project is essential for this project. We interact most intimately with Project 3 on chemistry (Smith) and Project 5 on virology (Sodroski), but also significantly with Project 2 on peptidomimetics and miniproteins (Chaiken), Project 4 on thermodynamics (Friere), and with both the computation core (LaLonde) and the protein core (Chaiken) [unreadable] [unreadable] [unreadable]